TARGETING A CIRCULAR RNA CDR1AS AS LOCUS ENHANCES INJURY-INDUCED REGENERATION OF THE INTESTINAL EPITHELIUM VIA MICRORNA DEREGULATION

The mammalian tissues express a huge number of circular RNAs (circRNAs) with an active role in gene regulation. circRNAs are covalently closed at the 5’ and 3’ ends, unusually stable, and are highly distributed in the cytoplasm. We recently reported that ~300 circRNAs, including Cdr1as, are differentially expressed in damaged intestinal mucosa in septic mice, but functions of most of these circRNAs in the intestinal epithelium remain unknown. In this study, we elucidated the in vivo function of the circRNA Cdr1as in the intestinal epithelium and identified Cdr1as as a repressor of mucosal regeneration. Methods: Cdr1as knockout (Cdr1as-/-) mice were generated by CRISPR-Cas9. Intestinal mucosa was collected from mice after injury and from patients with IBD and sepsis. Colonic mucosal injury was induced by 3% dextran sulfate sodium (DSS) in drinking water, while small intestinal mucosal injury was produced by cecal ligation and puncture or mesenteric ischemia/reperfusion (I/R). Primary enterocytes were isolated from the small intestine of mice and organoids were derived. Caco-2 cells were used for an in vitro intestinal epithelial injury model. Levels of Cdr1as were elevated in vitro and ex vivo by transfection with a lentiviral vector that expressed Cdr1as. Results: Colitis and septic stress increased the levels of intestinal mucosal Cdr1as in mice and human intestinal mucosa from patients with IBD and sepsis also exhibited increased Cdr1as abundance. After excluding the off-target genes, three founders of Cdr1as-/- mice were selected and shown to bear with a specific ablation of the Cdr1as locus. Loss of the Cdr1as locus from the mouse genome enhanced renewal of the intestinal mucosa, promoted injury-induced epithelial regeneration after exposure to I/R, and protected the mucosa against DSS-induced colitis. Cdr1as-/- mice also displayed deregulated expression of >40 microRNAs in the intestinal epithelium, including a significant decrease in miR-195 content. Increasing the levels of Cdr1as inhibited intestinal epithelial repair after wounding in vitro and repressed growth of intestinal organoids ex vivo but this inhibition was abolished by miR-195 silencing. Expression of miR-195 was posttranscriptionally misregulated in Cdr1as-/- mice by altering the stability of the miR-195 precursor (pre-miR-195) and its biogenesis. The half-life of pre-miR-195 increased in cells overexpressing Cdr1as, whereas the levels of Dicer complex proteins (essential for miRNA processing in the cytoplasm) decreased in the Cdr1as-deficient intestinal epithelium. Conclusions: These findings indicate that Cdr1as downregulates mucosal regeneration after injury and impairs epithelial defense via interaction with miR-195 and highlight a novel role of increased Cdr1as in the pathogenesis of chronic and unhealed wounds and disrupted renewal of the intestinal mucosa.